Erectile dysfunction, the inability of the penis to [attain or maintain] a rigid erection, is associated with aging. In the aging rat model, a defective relaxation of the penile corpora cavernosal smooth muscle results from a loss of tissue compliance, [and/or, possibly excessive sympathetic tone,] and/or, 61 reduced synthesis of nitric oxide (NO), the main mediator of penile erection. Our laboratory found and cloned PnNOS, a novel variant of neuronal nitric oxide synthase (nNOS), and showed it is the only nNOS expressed in the rat penis [both as full- length and shorter protein variants. A putative endothelial NOS (eNOS) variant was also detected. We have also shown that a protein Inhibitor of NOS activity (PIN), is expressed in the rat and human penis.] We also found that the aging-associated erectile dysfunction in the rat can be ameliorated by gene therapy with cDNA constructs of penile inducible NOS (iNOS). The aims of this project are first to determine whether aging affects the expression pattern of PnNOS and NOS (eNOS) variants in the penis by western and northern blots, RT/PCR, immunocytochemistry and in situ hybridization, and whether the reduction in penile NOS enzyme activity in very old animals is caused by NOS binding to inhibitors or by NOS glycation. Gene therapy of the corpora cavernosa in aged rats with PnNOS Will then be investigated, using several regimes and cDNA constructs in conjunction with L-arginine, to stimulate NOS activity. The ability to increase the erectile response of aged rats to electrical field stimulation (EFS) of the cavernosal nerve, and penile reflexes, will be determined. [Finally, the effects of aging on the levels of PIN binding to PnNOS variants will be determined in the rat penis with immunodetection techniques. An anti-PIN gene therapy with the cDNA for the truncated PnNOS unable to bind PIN (PnNOSbeta) will be tested to stimulate PIN-independent NOS activity. Alternatively, the cDNA for the nNOS protein domain that binds PIN will be given to quench PIN effects, and antisense PIN oligonucleotides will be investigated for a possible down-regulation of PIN translation. Other putative protein modulators of PnNOS activity will be searched for using the random display peptide library]. The long-term goal is to validate pre-clinically the use of these agents for medical treatment of erectile dysfunction associated with aging.